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Predicting transition temperatures of elastin-like polypeptide fusion proteins.

ABSTRACT: Elastin-like polypeptides (ELPs) are thermally sensitive peptide polymers that undergo thermally triggered phase separation and this behavior is imparted to soluble proteins when they are fused to an ELP. The transition temperature of the ELP fusion protein is observed to be different than that of a free ELP, indicating that the surface properties of the fused protein modulate the thermal behavior of ELPs. Understanding this effect is important for the rational design of applications that exploit the phase transition behavior of ELP fusion proteins. We had previously developed a biophysical model that explained the effect of hydrophobic proteins on depressing the transition temperature of ELP fusion proteins relative to free ELP. Here, we extend the model to elucidate the effect of hydrophilic proteins on the thermal behavior of ELP fusion proteins. A linear correlation was found between overall residue composition of accessible protein surface weighted by a characteristic transition temperature for each residue and the difference in transition temperatures between the ELP protein fusion and the corresponding free ELP. In breaking down the contribution of residues to polar, nonpolar, and charged, the model revealed that charged residues are the most important parameter in altering the transition temperature of an ELP fusion relative to the free ELP.

SUBMITTER: Christensen T

PROVIDER: S-EPMC3667497 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Predicting transition temperatures of elastin-like polypeptide fusion proteins.

Christensen Trine T Hassouneh Wafa W Trabbic-Carlson Kimberley K Chilkoti Ashutosh A

Biomacromolecules 20130408 5

Elastin-like polypeptides (ELPs) are thermally sensitive peptide polymers that undergo thermally triggered phase separation and this behavior is imparted to soluble proteins when they are fused to an ELP. The transition temperature of the ELP fusion protein is observed to be different than that of a free ELP, indicating that the surface properties of the fused protein modulate the thermal behavior of ELPs. Understanding this effect is important for the rational design of applications that exploi ...[more]

PMID: 23565607

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Project description:BACKGROUND: Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. RESULTS: The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. CONCLUSION: The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels of the recombinant protein and induce the formation of PBs regardless of the cultivar used. However, a specific level of recombinant protein accumulation needs to be reached for PBs to form.

Project description:BackgroundIn the enzymatic conversion of biomass, it becomes an important issue to efficiently and cost-effectively degrade cellulose into fermentable glucose. β-Glucosidase (Bgluc), an essential member of cellulases, plays a critical role in cellulosic biomass degradation. The difficulty in improving the stability of Bgluc has been a bottleneck in the enzyme-dependent cellulose degradation. The traditional method of protein purification, however, leads to higher production cost and a decrease in activity. To simplify and efficiently purify Bgluc with modified special properties, Bgluc-tagged ELP and His with defined phase transitions was designed to facilitate the process.ResultsHere, a novel binary ELP and His tag was fused with Bgluc from termite Coptotermes formosanus to construct a Bgluc-linker-ELP-His recombinant fusion protein (BglucLEH). The recombinant plasmid Bgluc expressing a His tag (BglucH) was also constructed. The BglucLEH and BglucH were expressed in E. coli BL21 and purified using inverse transition cycling (ITC) or Ni-NTA resin. The optimum salt concentration for the ITC purification of BglucLEH was 0.5 M (NH4)2SO4 and the specific activity of BglucLEH purified by ITC was 75.5 U/mg for substrate p-NPG, which was slightly higher than that of BglucLEH purified by Ni-NTA (68.2 U/mg). The recovery rate and purification fold of BglucLEH purified by ITC and Ni-NTA were 77.8%, 79.1% and 12.60, 11.60, respectively. The results indicated that purification with ITC was superior to the traditional Ni-NTA. The K m of BglucLEH and BglucH for p-NPG was 5.27 and 5.73 mM, respectively. The K ca t/K m (14.79 S-1 mM-1) of BglucLEH was higher than that of BglucH (12.10 S-1 mM-1). The effects of ELP tag on the enzyme activity, secondary structure and protein stability were also studied. The results showed that ELP tag did not affect the secondary structure or enzyme activity of Bgluc. More importantly, ELP improved the protein stability in harsh conditions such as heating and exposure to denaturant.ConclusionThe Bgluc-linker-ELP-His system shows wide application prospect in maintaining the activity, efficient purification and improving the stability of Bgluc. These properties of BglucLEH make it an interesting tool to reduce cost, to improve the efficiency of biocatalyst and potentially to enhance the degradation of lignocellulosic biomass.

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Predicting transition temperatures of elastin-like polypeptide fusion proteins.

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Predicting transition temperatures of elastin-like polypeptide fusion proteins.

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